Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects about 290 million people and kills ~900,000 annually. Therapy primarily employs nucleos(t)ide analogs that target the viral DNA polymerase (P), but only a few percent of patients clear the virus so therapy is life-long. Reverse transcription is catalyzed by coordinate action of the viral DNA polymerase (RT) that synthesized the DNA and the ribonuclease H (RNaseH) that destroys the RNA after it has been copied into DNA. The RT and RNaseH comprise adjacent domains of P, and the 2 enzymatic activities are allosteric and/or kinetically linked during replication. Newly synthesized HBV genomes have 3 fates: to become nuclear covalently closed circular DNA (cccDNA) molecules, be secreted within virions, or be integrated into the cellular genome. P is also a regulatory protein that accumulates at the endoplasmic reticulum and helps suppress interferon responses. The role of the RT in HBV replication is fairly well understood, but very little is known about how the RNaseH contributes to HBV biology. We recently expressed recombinant HBV RNaseH suitable for mechanistic analyses and drug discovery for the first time. We then developed the first screening pipeline for HBV RNaseH inhibitors and identified >130 compounds that block HBV replication by inhibiting the RNaseH. The recombinant RNaseH and inhibitors are unique new tools to probe contributions of the RNaseH to viral biology. Premise: Our recombinant HBV RNaseH, RNaseH inhibitors, and RNaseH assays enable studies to reveal how the RNaseH contributes to viral biology and how the enzyme can evolve resistance to RNaseH inhibitors. Aim 1. What are the effects of inhibiting the RNaseH on HBV reverse transcription? We will evaluate how RNaseH inhibitors affect RNA encapsidation, the fate of the RNA during reverse transcription when the RNaseH is inhibited, reversibility of damage to the viral genome induced by RNaseH inhibitors, how blocking the RNaseH affects cccDNA synthesis, and the specific infectivity of virions made without RNaseH activity. Aim 2. What is the potential for resistance to RNaseH inhibitors? We will select resistance mutations to RNaseH inhibitors in cell culture and then define their effects on viral fitness and selectivity against inhibitors from 3 chemotypes. Aim 3. How does inhibiting the RNaseH affect capsids and HBV?s interaction with cells? We will define how RNA:DNA heteroduplexes generated by inhibiting the RNaseH affect HBV capsids, the effects of RNA:DNA heteroduplexes induced by RNaseH inhibitors on interferon responses, and how heteroduplexes made without RNaseH activity affect integration of HBV DNA into the host genome. These data will define how the HBV RNaseH contributes to viral biology. This information will deepen our understanding of HBV?s interaction with cells and provide essential context for our ongoing development of RNaseH inhibitors as novel drugs intended to improve therapy for chronically infected patients.